Hypoxia is a well-characterized driver of solid tumor metastasis, and recent clinical evidence in the blood cancer multiple myeloma (MM) has uncovered a major role for hypoxia in MM disease progression. Patients with a more hypoxic MM phenotype exhibit substantially worse outcomes - a phenomenon observed in a wide range of solid tumors. In preclinical models of MM, hypoxia increases with disease progression leading to up- regulation of biomarkers (e.g., SNAIL, HIF-1a and 2, CXCR4) associated with increased aggressiveness and dissemination of tumor cells. Accordingly, our preliminary measurements of hypoxia using an exogenous hypoxia biomarker (pimonidazole) have confirmed that essentially all MM cells in established bone marrow (BM) lesions are hypoxic. Given that hypoxia can propel metastasis and increase resistance to chemotherapy in solid tumors, and that hypoxia levels correlate with MM disease progression, altering the hypoxic environment by delivering oxygen to the hypoxic tissue may improve MM disease progression and reduce treatment resistance. We have engineered H-NOX oxygen-carrying proteins that are optimally designed for tumor penetration and have an excellent safety profile. We therefore propose to use these long-acting and safe H-NOX oxygen carriers to suppress hypoxia in a disease model of MM in a manner that is not toxic to humans. By oxygenating the hypoxic MM lesions, this H-NOX oxygen carrier is likely to delay or inhibit the biological changes that contribute to MM progression, and may also sensitize MM cells to conventional chemotherapeutic treatments. Omniox has engineered the H-NOX oxygen-binding protein variants specifically to release oxygen in hypoxic niches. The initial effort (funded by SBIR NCI Phase I, II, and IIB awards) focused on developing OMX- 4.80 for the treatment of solid tumor hypoxia and enhancing radiation therapy. Because OMX-4.80 is designed to work over a period of hours, it is not suitable for clinical indications requiring sustained tumor oxygenation, such as MM. To address this, we have now engineered a second generation of H-NOX oxygen carriers with longer circulation half-lives (40-50 h) that maintain the ability to penetrate tumors and deliver oxygen over many weeks. Here, we propose to select an optimal H-NOX candidate that effectively blunts hypoxia-driven disease progression in mouse models of MM as a first step towards its clinical development. In this proposal, long-lived H-NOX oxygen carriers will be assessed for optimal hypoxia reduction in the BM niche of MM cells. We are collaborating with UCSF's MM Translation Initiative that was expressly established for this type of translational effort and has deep expertise in relevant MM disease models. We will employ MM1.s and RPMI-8826 human cell models to recapitulate hallmarks of clinical MM, including homing to the bone marrow, osteolytic complications, and eventual disease dissemination. Once we select an effective H- NOX variant in single dose Aim 1 studies, we will evaluate in Aim 2 a 2-week dosing regimen for its ability to alter the hypoxia-mediated disseminating phenotype. Finally, in Aim 3, using insights about hypoxia reduction effectiveness and its capacity to alter MM tumor cell phenotype, we will combine H-NOX with standard MM chemo and targeted therapies to evaluate chemosensitization and increase in efficacy. A successful outcome of this Phase I project will lead to refined dosing strategies, mechanism of action analyses and toxicological studies in support of IND filing and clinical development that will be outlined in a Phase II proposal.